The broad objective of this grant proposal is to study the mechanisms responsible for the fidelity of DNA synthesis. DNA polymerases are the key enzymes involved in replication and repair of DNA. An analysis of how polymerases control fidelity is central to understanding the biochemical basis of a wide variety of genetic diseases. Lesch-Nyhan syndrome and ADA deficiency are two examples of inherited childhood diseases that can arise from a single point mutation. Activation of oncogenes and inactivation of tumor suppressor genes leading to cancer can result from single base changes in DNA. Genetic defects in post-replication mismatch repair of DNA polymerase errors are a root cause of hereditary nonpolyposis colin cancer, along with a variety of other types of cancer. Previous fidelity studies have focused on individual DNA polymerases in the absence of polymerase accessory proteins required to sustain processive synthesis. This grant investigates the fidelity of purified procaryotic and eucaryotic DNA polymerase holoenzymes, pol III and pol II from Escherichia coli, and pol delta from Schizosaccharomyces pombe. A thorough understanding of fidelity mechanisms of DNA polymerases requires an analysis of the effects of sequence context and replication assessory proteins on fidelity. The proposed experiments, which include a full complement of polymerase subunits, are among the first of its kind, and make use of a mathematical model of polymerase fidelity and a gel fidelity assay that we've developed previously. The model is used to predict the effect of polymerase processivity subunits on base substitution fidelity. These predictions will be tested experimentally. Steady state kinetic experiments are designed to investigate the biochemical basis of mutational "hot" and "cold" spots. We propose to test the importance of hydrogen bonds between Watson-Crick base pairs on polymerase fidelity, by measuring the effects of base stacking on polymerase fidelity, in the absence of hydrogen bonding. Presteady state kinetic experiments, using fluorescent nucleotide analogs, are proposed to measure switching between polymerase and exonuclease active sites in "real-time". A second set of presteady state experiments are designed to determine the mechanism for loading and unloading the polymerase processivity clamp subunit onto DNA and to analyze the requirements for ATP hydrolysis for each step in the clamp-loading pathway. This pathway is required for Okazaki fragment formation during discontinuous lagging-strand DNA synthesis.